Escherichia coli vaccine

ABSTRACT

According to the present invention it has been found that a novel E. coli toxin or an immunogenic fragment thereof can be used in the preparation of vaccines for warm-blooded animals, and in particular for birds. Said toxin is found to be associated in flagellar structures attached to the bacteria, and these flagella or the free toxins can, after inactivation be used to immunize animals against E. coli infections.

This is a continuation of U.S. Ser. No. 07/967,912, filed Oct. 28, 1992,now abandoned, which is a file wrapper continuation of U.S. Ser. No.07/711,128, filed Jun. 6, 1991, now abandoned, which is a file wrappercontinuation of U.S. Ser. No. 07/562,527, filed Aug. 3, 1990, nowabandoned.

The invention is concerned with a vaccine for the protection ofindividuals against Escherichia coli (E. coli) infection, a toxin foruse in such a vaccine and a method for the purification of such a toxin.

E. coli is a widespread bacterium that colonizes the digestive tract ofmost animals. In general, such a colonization goes without seriousnegative effects--in most cases the bacterium even contributes toprocesses which are favourable to its host. However, occasionally E.coli causes serious diseases particularly in young animals. This canalso occur in birds and in the commercial poultry breeding such aninfection can become epidemic, leading to serious weakening or evenmassive mortality among the young birds.

Naturally, it has been attempted to have such E. coli infections amoungpoultry in hand by vaccination programs. To this end mature chickenshave been vaccinated with bacterins--inactivated E. coli bacteria (AvianDiseases 29(4), 1108-17 (1985)). A disadvantage of bacterin vaccines isthe concommitant serious side reactions. Furthermore, bacterinvaccination results primarily in antibodies against lipopolysaccharideswhich are only specific for a certain E. coli O serotype and hence arenot protective against other E. coli serotypes.

For the combatment of E. coli infections also frequently use is made ofvaccines based on pili obtained from these bacteria. However, thesevaccines only lead to a limited protection of not more than about 80% ofthe vaccinated individuals. For this reason E. coli vaccines oftencontain as a component yet another virulence factor: inactivated toxinof E. coli.

Many types of E. coli contain flagella, having a function in locomotion.For E. coli flagella have not been considered as a factor of virulence,and hence have not been included in E. coli vaccines.

According to the present invention, it has been found that the flagellaof E. coli are associated with a profound toxic activity towards Verocells, which was hitherto not recognized, and that these flagellartoxins are a significant factor of virulence.

In view of this finding vaccines have been prepared derived fromflagella of E. coli. According to the present invention whole flagellaof E. coli can be used, as well as substructures thereof composing saidflagella, e.g. flagellins or fragments or aggregates of the flagellinswhich protect individuals vaccinated therewith against E. coliinfections.

In experiments with a large number of E. coli strains, isolated mainlyfrom chicken but also from other animals and humans, flagella were foundto be associated with toxicity against Vero cells; this toxic activitywas found to be neutralized by antibodies against the flagella. Itfurther turned out that the toxicity of the flagella of all E. colistrains studied could be neutralized by a single antiserum raisedagainst flagella of one of the strains.

In view of this finding it is anticipated that vaccination with flagellaobtained from a single E. coli strain will provide protection againstinfection with all flagella bearing E. coli strains.

In view of the above considerations the vaccine according to theinvention is based on a novel class of toxins found in E. coli and whichare characterized in that they are of protein nature, are foundassociated in and/or with flagella, and have a molecular weight ofbetween 30-100 kD measured in SDS-PAGE, do not possess boundcarbohydrate residues, are toxic to Vero cells and to day-old chicks andkeep this toxicity even on heating for 1 hour at 100° C.

The combined characteristics distinguish these novel toxins from the E.coli toxins known in the art.

The above-described toxins are found among a great number of E. colistrains and are named here flagellar toxins (FT) because of theirstriking occurence in flagellar structures. These flagella generally aresignificantly larger than normal fimbriae (which typically are about 7nm in diameter and up till about 1 μm long), and are up to 25 nm thickand 7 μm long.

One member of the class of toxins according to the present invention wasisolated from the chicken E. coli strain CH7 (015:K14:H10) according tothe procedure outlined in Example 1. This CH7-FT can be isolated inseveral forms: either associated as the native flagella of type H10, oras the free toxin, or re-associated to small needle-like filamentsobtained from the free toxin. Typical characteristics of this CH7-FT ontop of the afore-mentioned general characteristics are a subunitmolecular weight of about 47 kD as determined by SDS-PAGE, aniso-electric pH of about 4.8 and the partial amino-terminal amino acidsequence: Ala-Gln-Val-Ile-Asn-Thr-Asn-Ser-Leu-Ser-Leu-(?)-Thr-Gln. (Thecharacterization of CH7-FT is described in Example 2).

Antiserum raised against this CH7-FT was found to cross-react with FT ofall other E. coli strains tested (Example 3). Accordingly, such anantiserum against CH7-FT can be used to characterize all other FT'saccording to the present invention. Furthermore, monoclonal antibodieswere either specific or cross-reactive with FT of all other E. colistrains tested.

The present invention also comprises vaccines with immunizing activityagainst E. coli infection, wherein the active ingredient is aninactivated toxin according to the present invention.

Such a vaccine suitably contains said toxic flagella as these flagellacan readily be obtained by culturing E. coli bacteria under conditionspromoting the formation of flagella, and separating the flagella or thecell free supernatant from the bacteria. The FT can be further purifiedby removal of low molecular weight components of the supernatant usingultrafiltration and/or molecular sieve chromatography.

During this purification process the fraction enriched in FT can bemonitored by its reactivity with the monoclonal antibodies raisedagainst CH7-FT.

A vaccine according to the invention may also comprise a fragment of FTwhich protects individuals vaccinated therewith against E. coliinfection.

A FT to be incorporated into a vaccine according to the invention can beobtained by chemical synthesis, purification from E. coli cell cultureor by recombinant DNA technology.

In the latter case nucleic acid sequences encoding above-mentionedprotein or fragments thereof can for example be identified by screeninga genomic E. coli DNA bank for individual clones comprising saidsequences, e.g. by using a specific reaction with polyclonal ormonoclonal antibodies elicited against FT. The nucleic acid sequencescan be ligated to various expression effecting DNA sequences, resultingin a so called recombinant nucleic acid molecule which can be used forthe transformation of a suitable host. Such hybrid DNA molecules can forexample be derived from plasmids, phages or from nucleic acid sequencespresent in viruses. The host cell can be of prokaryotic origin, e.g.bacteria or eukaryotic origin such as mammalian cells. The transformedhost cells can be used to produce the FT whereafter said protein can beisolated and subsequently incorporated into a vaccine according to theinvention.

In another embodiment a live vector vaccine can be prepared comprisingnon-pathogenic micro-organisms, e.g. viruses or bacteria containing thegene encoding the FT.

Apart from FT a vaccine according to the present invention may alsocontain an aqueous medium or a water containing suspension, and/or otherconstituents e.g. in order to increase the activity and/or the shelflife. These constituents may be salts, agents to inactivate the toxicactivity of FT while maintaining its immunogenic properties (e.g.formalin), pH buffers, emulsifiers and adjuvants to improve the immuneresponse (e.g. mineral oils, muramyl dipeptide, aluminium hydroxide,saponin, polyanions and amphiphatic substances).

The vaccine is useful in immunizing warmblooded animals (including man)against E. coli infections and in particular can be used to combat E.coli infections in birds.

To this end the vaccine preferably is administered parenterally, forexample subcutaneously or intramuscularly. The vaccine may beadministered in this manner both for the active immunization of thevaccinated birds and to laying birds for the passive immunization of theoffspring thereof. In immunized laying birds, the antibodies raised inthem will, of course, be introduced into the yolks of their eggs andtherefore subsequently in the hatched chicks.

Both the composition of the vaccine and the vaccination system can bevaried and depend on the type of animal to be protected, the age and theweight of the animal, the desired duration of the protection, the methodof administration and on the question of whether active immunization orpassive immunization by means of maternal antibodies is desired. Theoptimally effective quantity of the active component in the vaccine isapproximately 10-100 μg per dose for parenteral vaccination of poultry.The vaccine may be combined with other relevant vaccines.

EXAMPLE 1 Isolation of Flagellar Toxin of E. coli strain CH7

A. Preparation of Flagellar Toxin

E. coli strain CH7 (015:K14:H10) was cultured overnight in a Biostat Efermentor (Braun) in 12 l Trypticase Soy Broth (B.B.L.) at a pO₂ settingof 14% and variable stirring from 100-500 rpm. The culture wasconcentrated to approx. 1 l in a Pellicon filter system (Millipore) witha HVLP filter. Bacteria were centrifuged for 30 minutes at 10,000 rpm(GSA rotor, Sorvall), the supernatant filtered through a 0.45 μm filterand added to the HVLP filtrate.

The filtrate was concentrated to approx. 1 l and washed with 3 times 1 l0.2 mol/l Tris HCl buffer using a PTTK filter.

The PTTK concentrate was repetitively eluted in portions of about110-160 ml over a sepharose 4B-Cl column (Pharmacia, Uppsala Sweden)with a height of 10 cm and an area of 154 cm² (Amicon model P140×250)equilibrated in phosphate buffer 50 mmol/l pH 7.2 with 0.1% NaN₃ as apreservative (PB). The column was eluted with PB until the baseline ofthe recorder was zero again.

The fractions of the first peak, containing the high molecular weightmolecules were pooled, and concentrated on an YM-100 ultrafiltrationfilter (.O slashed. 62 mm Amicon, with Amicon UF model 202) until theprotein concentration was about 2-3 mg/ml. The concentrate was dialysedtwice against 5 l Tris HCI buffer 20 mmol/l pH 7.5 with 0.1% NaN₃ aspreservative.

B. Preparation of Free Toxin

The concentrate was preparatively electrophoreted by the method ofLaemmli (Nature 227, 680-4; 1970). It was 1:1.67 diluted in samplebuffer composed of 20 ml glycerol, 20 ml Tris-HCl buffer 0,5 mol/l pH6.8, 20 ml 10% SDS, 5 ml 2-mercaptoethanol (ME) and 2 ml 0.05%bromophenol blue.

Then it was boiled for about 5 minutes in water, cooled off andelectrophoreted on a 12% (acrylamide:bis 30:0.8) preparativepolyacrylamide slab gel of 16×0.6 cm. Typically sample loads were about15-23 mg protein per gel (7.5 ml concentrate and 5.0 ml sample bufer).The electrophoreses was performed on a Protean cell model 1423 (Bio-Rad,Richmond USA) or a model SE600 (Hoefer, San Francisco, USA). After thefront was eluted from the gel the electrophoresis was continued foranother hour at 200 V. The gel was cut in slices of about 1.5-2 mm.

The proteins were eluted in 10 ml 0.89% sodiumchloride solution+0.1%NaN₃, for three hours at room-temperature and overnight at 4° C. undercontinuous agitation. The slices were removed and the solutions werefiltrated over a 0.45μfilter. The fractions containing pure toxinsubunits were pooled, and stored at -20° C.

The protein content in the samples was measured by a modifiedFolin-Ciocalteu assay (J. Biol. Chem. 73, 627; 1927), polysaccharide wasmeasured with use of the phenol-sulphuric acid assay according to Dubois(Anal. Chem. 28, 350-6; 1956).

EXAMPLE 2 Characterization of Toxin of E. coli strain CH7

A. Letality for One Day Old Chickens

In a first experiment 0.2 and 0.5 ml of various E. coli toxinpreparations were injected IP into one-day-old SPF broiler chickens(GVP, Doorn). In a second experiment 0.5 ml of toxin preparations wereinjected IV into broilers of 3 weeks old. Deaths were recorded for 7days after injection.

Results

As shown in Table 1, toxin preparations from both chicken E. colistrains CH2 and CH7 were lethal for one-day-old chickens after IPinjection. For strain CH7 both supernatant as well as lysate were toxic,whereas for strain CH2 especially the lysate was toxic.

                  TABLE 1                                                         ______________________________________                                        Letality for one-day-old chickens: IP injection                               of supernatant or lysate from E. coli strains.                                                    Dead chickens/total                                       Preparation  Dosis  injected on day                                           injected*    (ml)   1          4    7                                         ______________________________________                                        Sterile TSB  0.5                    1/10                                      ZF24 sup     0.2                    1/10                                      sup          0.5                    1/10                                      lys          0.2                    1/10                                      lys          0.5                    1/10                                      CH2 sup      0.2               1/10 1/10                                      sup          0.5               1/10 1/10                                      lys          0.2    9/10       9/10 9/10                                      lys          0.5    6/10       7/10 8/10                                      CH7 sup      0.2    2/10       4/10 5/10                                      sup          0.5    4/10       5/10 5/10                                      lys          0.2    4/10       4/10 4/10                                      lys          0.5    7/10       7/10 7/10                                      ______________________________________                                         *Strains CH2 and CH7 are chicken E. coli isolates; Strain ZF24 is an          avirulent E. coli isolate of human feces.                                

IV injection of similar preparations into 3 weeks old chickens had noeffect at all (data not shown).

B. Vero Test

Vero cells were grown at 37° C. in a 5% CO₂ atmosphere in medium 6 (perliter containing 85 ml MEM Eagle, 100 ml tryptose phosphate broth, 50 ml4.4% NaHCO₃) supplemented with 5% Fetal Calf Serum (FCS) and 200 U/mlpenicillin and 200 μg/ml streptomycin, and after filter sterilisationsupplemented with 2 μg/ml fungizone. After trypsinisation the cells wereseeded into 96-wells flatbottom polystyrene culture plates (Greiner)with 200 μl per well of complete medium 6 containing 2×10₅ cells per ml.After overnight incubation monolayers are established. The medium wasdiscarded and replaced by 200 μl per well of medium 6 without FCS butsupplemented with 10 μg/ml xanthine (3-isobutyl-1-methyl-xanthine;Sigma). Subsequently, 20 μl per well of (serial dilutions of) toxinpreparations were added. The cytopathological effect (CPE) was recordedafter 5 days incubation.

Screening of strains for toxin production was performed firstly byadding 20 μl per well of undiluted and 1:2 diluted supernatants.Secondly, strains from which the supernatants were negative in the Verotest, were tested for intracellular toxin production by adding 50 μl perwell of undiluted and 1:2 diluted bacterial lysates.

Results

Initially, the strains listed in Table 2 were tested for toxinproduction. Some strains excreted toxin in the supernatant whereas withother strains the toxin was intracellular and/or only detectable afterultrasonic disruption of the bacterial cells. The cytopathologicaleffect was rounding and shrinking of the Vero cells, whereas themonolayer stayed intact in most cases.

                  TABLE 2                                                         ______________________________________                                        Vero cell toxicity of various E. coli strains                                                         toxin titer**                                                                           in                                          Strain*    Serotype     supernatant                                                                             lysate                                      ______________________________________                                        JA221      --           --        --                                          ZF24       023:K?:H--   --        --                                          CH1        078:K80      --        --                                          CH2        078:K80:H4   --        8                                           CH3        045:K--:H9    32       64                                          CH4        02:K1:H--    --        8                                           CH5        02:K1:H5      32       512                                         CH6        01:K1:H--    --        4                                           CH7        015:K14:H10  128       1,024                                       CH8        0115:K?      --        16                                          CH13       035:K--       32                                                   ______________________________________                                         *JA221 is an E. Coli K12 strain, ZF24 see Table 1; CH strains are chicken     isolates                                                                      **toxin titer is defined as the reciprocal of the last dilution giving a      toxic effect                                                             

C. Stability of the Toxin

Preliminary characterisation of the identified toxin was performed bytesting the sensitivity of toxin preparations for various treatments. pHsensitivity was tested by adjusting toxin to pH 3 to 10 andneutralisation after overnight incubation at room temperature, prior totoxicity testing.

For heat sensitivity testing toxin preparations were heated at varioustemperatures. The effect of SDS and ME was tested by heating toxin inthe presence of 1% SDS and of 1% SDS with 2.5% ME, and subsequentdialysing against saline. For testing the sensitivity for ureum, 6Mureum was added to toxin preparations for 1 hour and dialysed againstsaline.

Formalin sensitivity was tested by the addition of variousconcentrations of formalin, incubation overnight at varioustemperatures, and dialysing prior to toxicity testing in the Vero cellassay. The sensitivity to trypsin was tested by the addition of 100μg/ml trypsin (bovine pancreas; Millipore), incubation at 37° C. for 4hours, and subsequent addition of 150 μg/ml trypsin inhibitor (soybean;Sigma) for 30 min. at 37° C. prior to toxicity testing.

Results Since exact chicken toxin titer determinations in the Vero celltoxicity assay are not very reproducible due to variations in thecondition of the Vero cells on different days, results are presentedhere only as examples of typical experiments.

Treatment of CH5 and CH7 supernatant at pH 3 up to and including 10 didnot affect the toxicity, the toxin titers were invariable 32-64 and128-256 respectively.

The heat sensitivity and the sensitivity to SDS or SDS+ME treatment isshown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Effect on chicken E. coli toxin titers of                                     heating toxin preparations in the absence and                                 presence of SDS or SDS + ME                                                   Treatment      CH2 lys.   CH5 sup. CH7 sup.                                   ______________________________________                                        control        8          32       128                                        80° C. (1 h)                                                                          4          8        64                                         100° C. (1 h)                                                                         4          8        16                                         120° C. (20 min.)                                                                     0          0        0                                          65° C. (10 min.)   16       64                                         SDS, 65° C. (10 min.)                                                                            32       64                                         SDS + ME, 65° C. (10 min.)                                                                       32       64                                         100° C. (10 min.)  8        64                                         SDS, 100° C. (10 min.)                                                                           32       128                                        SDS + ME, 100° C. (10 min.)                                                                      16       128                                        ______________________________________                                    

Although the toxicity of CH2 lysate (lys.) and of CH5 and CH7supernatants (sup.) was somewhat decreased after prolonged exposure tohigher temperatures and abolished completely after heating at 120° C.,the toxin has to be considered as relatively heat-stable. Heating for 10min. in the presence of SDS or even SDS+ME had no effect on the toxicityof CH5 and CH7 supernatants.

Treatment of CH2 lysate and CH5 and CH7 supernatants with 6M ureum hadno effect at all on the respective VT titers.

As shown in Table 4, the toxicity of CH7 supernatant is inactivated byformalin at room temperature and at 37° C.

The toxicity of both CH5 and CH7 supernatants was abolished completelyafter treatment with trypsin, whereas sham treatment and treatment withtrypsin inhibitor alone had no effect at all on toxicity.

                  TABLE 4                                                         ______________________________________                                        Inactivation of CH7 supernatant toxicity by                                   incubation overnight with various concentrations                              of formalin                                                                   Formalin concentration                                                                        Toxin titer after incubation at                               (%)             room temp. 37° C.                                      ______________________________________                                        0               64         64                                                 0.2             32         16                                                 0.5             16         4                                                  1.0             8          2                                                  2.0             1          0                                                  ______________________________________                                    

D. Molecular Weight Determination

The molecular weight of the toxin of strain CH7 was determined byanalytical gelelectrophoresis in 12% gels (acrylamide:bis=30:0.8) by themethod of Laemmli by comparison with standards.

Gels were stained with coomassie-brilliant blue (CBB). Scans were madeusing a gelscanner model CS-930 and recorder DR-2 (Shimadzu, KyotoJapan).

FIG. 1 shows a scan after running the gel loaded with molecular weightmarkers, stained with coomassie-brilliant blue. The standardscorresponding with peak 1-6, have molecular weights of 78000, 66000,45000, 30000, 17200 and 12300 D, respectively (LKB 1860-12 Bromma,Sweden).

In FIGS. 2 and 3 are represented the scans of the products obtained fromstep A and step B of Example 1, respectively.

The molecular weight of the toxin subunit of E. coli strain CH7 wasfound in these experiments to be about 47 kD.

E. Iso-electric Point Determination

The iso-electric point of the toxin was determined focussing 3 ml toxinof E. coli strain CH7 obtained from step A of Example 1 together with amixture of 0.5 ml Servalytes pH 3-7 (analytical grade Serva HeidelbergGermany) and 46.5 ml aqua dest for 5 hours at 12 W Rotofor, Bio-RadRichmond USA). Toxin content was detected by analytical gelelectrophoresis.

The results of this experiment are summarized in table 5.

It was found from these results that the toxin of the E. coli strain CH7has an iso-electric point at about pH 4.8.

Results

                  TABLE 5                                                         ______________________________________                                        pH and toxin values after focussing of 3 ml                                   seph 4B-Cl sample.                                                            Fraction                                                                              pH       Toxin*  Fraction pH   Toxin*                                 ______________________________________                                        1       2.68     +       11       5.50 --                                     2       3.26     ±    12       5.82 --                                     3       3.50     -       13       6.23 --                                     4       3.73     -       14       6.57 --                                     5       4.18     -       15       6.85 --                                     6       4.38     ±    16       7.14 --                                     7       4.55     ++      17       7.47 --                                     8       4.80     ++++    18       7.97 --                                     9       5.09     ++      19       8.41 --                                     10      5.32     +       20       8.75 --                                     ______________________________________                                         * - = no toxin visible                                                        ± = just visible                                                           + = visible                                                                   ++, +++, ++++ = increasing amounts of toxin                              

F. Saccharide Contents

The toxin of E. coli strain CH7 obtained from step B. of Example 1 didneither contain polysaccharide nor any sugars as determined in thephenol-sulphuric acid assay of Dubois et al. (Analytical Chemistry 28,350-356; 1956). In the Limulus Amoebocyte Lysate test (Pyrotell, Mass.,USA) no significant LPS (endotoxin) activity was detected.

G. Amino Acid Analysis

The N-terminal amino acid sequence was determined by the liquid phaseDABITC procedure according to Chang (Methods Enzymology 91, 455-466;1983). Identification of DABTH-amino acids was performed by thin-layerchromatography. The amino acid composition was determined by the PTCtechnique as described by Janssen et al. (Chromatographia 22, 345-358;1986), with the assumption that the subunit molecular weight of 47 kDfor CH7-FT corresponds with a total of 446 amino acids.

Results

The toxin of E. coli strain CH7 obtained from step A and step B ofExample 1 had the following N-terminal amino acid sequence:

    Ala-Gln-Val-Ile-Asn-Thr-Asn-Ser-Leu-Ser-Leu-(?)-Thr-Gln

This sequence is identical to the N-terminal amino acid sequence of E.coli K-12 flagellin as described by Kuwajima et al. (Journal ofBacteriology 168, 1479-1483; 1986).

The amino acid composition of the toxin is given in Table 6 and alsoshows homology with E. coli K-12 flagellin to a considerable degree.

                  TABLE 6                                                         ______________________________________                                        Estimation of the amino acid composition of                                   CH7-FT and comparison with the amino acid                                     composition of E. coli K-12 flagellin: number                                 of amino acids per subunit (percentage).                                      Amino                      E. coli K-12                                       acid     CH7-FT.sup.1)     flagellin.sup.2)                                   ______________________________________                                        Ala             50(11.2)    59(11.9)                                          Arg             12(2.7)     11(2.2)                                           Asn                         48                                                                52(11.7)              (17.5)                                  Asp                         39                                                Cys             4(0.9)      0(0)                                              Gln                         27                                                                43(9.6)               (6.2)                                   Glu                         14                                                Gly             37(8.3)     44(8.9)                                           His             0(0)        0(0)                                              Ile             24(5.4)     28(5.6)                                           Leu             32(7.2)     37(7.4)                                           Lys             28(6.3)     25(5.0)                                           Met             2(0.4)      3(0.6)                                            Phe             9(2.0)      5(1.0)                                            Pro             8(1.7)      6(1.2)                                            Ser             58(13.0)    43(8.7)                                           Thr             48(10.8)    65(13.1)                                          Trp             0(0)        0(0)                                              Tyr             11(2.5)     10(2.0)                                           Val             28(6.3)     33(6.6)                                           Total          446         497                                                ______________________________________                                         .sup.1) Estimated by the PTC technique (Chromatographia 22, 345-358;          1986).                                                                        .sup.2) Calculated on the basis of the DNA sequence (Journal of               Bacteriology 168, 1479-1483; 1986).                                      

EXAMPLE 3 Screening of chicken E. coli Strains for FT Expression andSerological Characterization of FT

A total of 124 chicken E. coli isolates from all over the world werescreened for their toxicity, motility and expression of FT antigen onthe bacterial surface. Polyclonal and monoclonal antibodies were used tovisualize the FT and to investigate cross-reactions.

Methods

Toxicity Testing and Toxin Neutralization

The strains were tested for toxicity on Vero cells as described inExample 2B. For neutralization, toxin preparations were incubated withantiserum dilutions for 2 h at 37° C. prior to toxicity testing.

Motility Testing

Motility of the strains was tested in U-shape tubes containing nutrientbroth with low (0.25%) agar concentration. These U-tubes were inoculatedwith an E. coli strain at one side, and migration to the other end ofthe tube was recorded after overnight incubation at 37° C.

Antisera Production

Antisera were raised in rabbits and chickens against the FT of E. colistrain CH7 prepared as described in Example 1A. The toxins of strainsCH5 and CH7 prepared as described in Example 1B were used for theproduction of monoclonal antibodies (MoAb). For MoAb production spleencells from immunized mice were fused with myeloma cells and theresulting hybridomas were screened for anti-toxin antibody secretion inan ELISA. Positive hybridomas were cloned by limiting dilution. Asciticfluid was prepared by intraperitoneal injection of cloned hybridomasinto mice. Ascites was inactivated at 56° C. for 10 min., lipids wereextracted with 1,1,2-trichlorotrifluoroethane (Merck), and MoAbs wereprecipitated with 50% saturated ammonium sulphate.

FT Antigen Expression by E. coli Strains

Rabbit antiserum raised against the FT of strain CH7 (015:K14:H10) wasabsorbed for 24 h at room temperature with the non-toxigenic E. colistrain RDEC-1 (015:K14). This absorbed antiserum was used to screenstrains for FT expression in a whole bacteria ELISA carried out asfollows.

Bacteria were grown for 6 hours in TSB without agitation, spun down at3,000 rpm for 15 min. (Sorvall RT6000) and resuspended in CBB buffer(1.59 g/l Na₂ CO₃ ; 2.93 g/l NaHCO₃ ; 0.2 g/l NaN₃ ; pH 9.6) to an O.D.at 660 nm of 0.140-0.180. Flatbottom polystyrene microtiterplates(Greiner) were seeded with 100 μl per well of these bacterialsuspensions and allowed to dry up at 50° C. overnight. The plates werewashed with tap water and blocked for 1 h. at room temperature with 110μl per well of PBS-T-N (0.04M PBS; pH 7.2; 0.5% Tween 80; 15% NewbornCalf Serum). Subsequently 100 μl per well of serial dilutions ofabsorbed serum were added, diluted in PBS-T-N and starting with a 1:100dilution. Two wells per strain with PBS-T-N served as backgroundcontrols. After 1 h. incubation at 37° C., the plates were washed and100 μl per well of peroxidase-conjugated goat-anti-rabbit IgG(H+L) wasadded to each well in the appropriate dilution in PBS-T-N. Afterincubation at 37° C. for 30 min. the plates were washed again. Antibodybinding was detected calorimetrically by adding 100 μl per well ofTMB-substrate buffer, containing ureum-peroxide (Organon Teknika, Oss)and 3,3',5,5'-tetramethylbenzidine in sodium acetate-citric acid buffer(pH 5.5). The reaction was developed in the dark for 10 min., stopped byadding 50 μl 4N H₂ SO₄, and measured in a Microelisa reader at 450 nm.Titers were determined as the highest antiserum dilution giving an A₄₅₀of at least 2 times the background A₄₅₀.

In each assay strains CH7 and RDEC-1 were included as positive andnegative controls respectively.

Western Blotting

Immunoblotting or Western blotting was performed essentially asdescribed by Muilerman et al. (Anal. Biochem. 120, 46-51; 1982). CrudeFT preparations of strains were prepared by growing bacteria inTrypticase Soy Broth for 6 h with agitation. Bacteria were removed bycentrifugation after vigorous mixing, and supernatant was concentratedapprox. 40 times by ethanol precipitation (1 part supernatant with 2parts 96% ethanol, overnight incubation at 4° C., centrifugation anddissolving the precipitate in 0.04 mol/l PBS, pH 7.2). These crude FTpreparations were run in SDS-PAGE and transblotted to cellulose nitratemembrane filter. Antigens were visualized by the successive incubationwith antibodies, appropriate peroxidase-conjugated anti-species IgG(H+L), and ureum peroxide with 3,3'-diaminobenzidine. 4HCl.

Immunogold-Electronmicroscopy (IG-EM)

IG-EM was carried out essentially as described by van Alphen et al.(Infect. Immun. 56, 1800-6; 1988). Briefly, bacteria grown in TrypticaseSoy Broth were incubated with antibody dilutions in PBS plus 1% BSA plus0.05% Tween 20 (PBS-B-T), washed thrice with PBS and incubated withprotein A labeled with gold spheres in PBS-B-T. After three morewashings with PBS bacteria were transferred to Formvar-coated grids andnegatively stained with 1% uranyl acetate or phosphotungstic acid.

Results

In a collection of 124 chicken E. coli strains from all over the world,73 strains (59%) excreted detectable amounts of toxin active on Verocells. A further 37 strains (30%) were toxic for Vero cells after lysisof the bacteria. In whole bacteria ELISA 52 strains (42%) reacted withantiserum raised against CH7-FT. All strains that were positive in theELISA also produced extracellular Vero toxin (Table 7).

                  TABLE 7                                                         ______________________________________                                        Relation between Vero toxicity and reactivity                                 with antiserum raised against CH7-FT (numbers                                 of strains)                                                                                    Vero toxicity.sup.2)                                                          +    -                                                       ______________________________________                                        anti CH7-FT                                                                              +           52      0                                              reactivity.sup.1)                                                                        -           21     51                                              ______________________________________                                         .sup.1) Reaction in whole bacteria Elisa with CH7FT antiserum                 .sup.2) Toxicity for Vero cells of bacterial culture supernatant.        

A strong correlation was found between Vero toxin excretion and motilityof the strains (Table 8).

                  TABLE 8                                                         ______________________________________                                        Relation between Vero toxicity and motility                                   (numbers of strains)                                                                           Vero toxicity.sup.2)                                                          +    -                                                       ______________________________________                                        Motility.sup.1)                                                                         +            69     10                                                        -             4     41                                              ______________________________________                                         .sup.1) Motility in Utubes                                                    .sup.2) Toxicity for Vero cells of bacterial culture supernatant.        

These results provide additional evidence that the Vero toxic activityresides in the flagella. It was also found that Vero toxicity increasedafter passage of bacteria through U-tubes. Furthermore, these resultsshow that the toxins of different strains are serologically crossreactive.

The cross reaction between the toxins of different strains was furtherinvestigated. In Table 9 it is shown that both rabbit and chickenantisera raised against FT of strain CH7 neutralized the Vero toxicityof all other toxigenic strains tested. MoAbs raised against FT ofstrains CH5 and CH7 did not neutralize toxicity at all.

                  TABLE 9                                                         ______________________________________                                        Neutralization of Vero toxicity in culture                                    supernatant by antisera raised against CH7-FT                                               Vero toxin titer.sup.1)                                                                        K07577.sup.3)                                                                        BB1-2.sup.4)                            Strain   Serotype   Control.sup.2)                                                                           (1:10) (1:10)                                  ______________________________________                                        CH3      O45:K--:H9 8          --     --                                      CH5      O2:K1:H5   16         --     --                                      CH7      O15:K14:H10                                                                              32         --     --                                      CH125    O1:K1:H7   32         4      --                                      CH135    O2:K1:H4   64         4      --                                      ______________________________________                                         .sup.1) see Table 2.                                                          .sup.2) Shamtreated supernatant, or treated with preimmune serum.             .sup.3) Rabbit anti CH7FT antiserum, 1:10 diluted.                            .sup.4) Chicken anti CH7FT antiserum, 1:10 diluted.                      

                                      TABLE 10                                    __________________________________________________________________________    Western blotting of crude FT preparations from                                various strains with antisera, and comparison                                 with corresponding flagellin molecular weight.                                           Antibodies.sup.1)    Flagellin                                     Strain                                                                            Serotype                                                                             KO7577                                                                             BB1-2                                                                             αH10                                                                       Int 1-7                                                                           Int 12-13                                                                          MW                                            __________________________________________________________________________    CH3 O45:K--:H9                                                                           70.sup.2)                                                                          70  70 --  70   69.sup.3)                                     CH5 O2:K1:H5                                                                             43   43  43 --  43   46.sup.3)                                     CH7 O15:K14:H10                                                                          47   47  47 47  47   45-47.sup.4)                                  CH125                                                                             O1:K1:H7                                                                             60   60  60 --  60   61.sup.3)                                     CH135                                                                             O2:K1:H4                                                                             35   35  35 --  35   37.sup.3)                                     __________________________________________________________________________     .sup.1) KO7577 = rabbit antiserum raised against CH7FT; BB1-2 = chicken       antiserum raised against CH7FT; αH10 = agglutinating antiserum for      H10 flagella typing, purchased from RIVM (Bilthoven); Int1-7 = MoAb raise     against CH7FT; Int12-13 = MoAb raised against CH5FT.                          .sup.2) Data represent approx. apparent MW of single or major bands in        blot in kD.                                                                   .sup.3) A. M. Lawn (J. Gen. Microbiol. 101, 112-130; 1977).                   .sup.4) Own observation with H10 flagella reference strains.             

The results of Western blotting of crude FT preparations with variousantisera are shown in Table 10. Rabbit and chicken antisera raisedagainst CH7-FT (KO7577 and BB1-2, respectively) reacted with all otherFT preparations tested, although the MW of the bands differed amongstrains. Identical results were obtained using an anti-H10-flagellaagglutinating antiserum. Also MoAb Int12-13, raised against CH5-FTshowed an identical pattern in Western blotting. MoAb Int1-7, raisedagainst CH7-FT, only reacted with the 47 kD band of CH7-FT. Strikingly,the flagellin MWs corresponding with the H types of the various strainswere almost identical with the apparent MWs of the respective FTs. Anumber of strains with H10 type flagella, obtained from RIVM(Bilthoven), showed bands at either 45 kD or 47 kD in Western blottingwith the polyclonal antisera. MoAb Int1-7 only reacted with the 47 kDband of H10 flagella strains. The intensity of the bands in Westernblotting was increased when strains were passed through U-tubes prior topreparing crude FT.

In IG-EM, flagella-like filaments on both CH5 and CH7 bacteria werelabeled with gold spheres, using polyclonal antisera raised againstCH7-FT. With MoAb Int1-7, raised against CH7-FT, only flagella-likefilaments on CH7 and not on CH5 bacteria were labeled with gold spheres.With MoAb Int12-13, raised against CH5-FT prepared as described inExample 1B using preparative SDS-PAGE, flagella-like filaments were notlabeled significantly; only some gold spheres were observed on CH5 andCH7 bacterial surfaces. In fact, MoAb Int12-13 only reacted withdissociated FT (Western blot, ELISA) and not with intact FT (IG-EM,ELISA).

All these results point out that the Vero toxic activity resides in theflagella, or that FT is identical to flagella. Furthermore, the FTs ofdifferent strains are serologically highly cross-reactive and alsocross-neutralizing.

EXAMPLE 4 Protection of Broilers by Passive Immunization

Antiserum was raised against CH7-FT by vaccinating chickens with CH7-FTprepared as described in Example 1A. Antisera from different chickenswere pooled and inactivated at 56° C. for 10 min.

Three-week old broilers (Euribrid, Boxmeer, The Netherlands) wereinjected intravenously with 1 ml of this CH7-FT antiserum. Within 1 hourafter antiserum injection the chickens were infected by injection of 0.2ml of bacterial suspension into the right posterior thoracic air sac.Bacteria were cultured overnight on blood agar base plates (Oxoid),suspended in PBS and diluted to the appropriate concentration. The E.coli strains used were all isolated from affected hearts of chickenswith colibacillosis. Control chickens, to which no antiserum wasadministered or only negative control serum, were infected with the samedoses of bacteria. After challenge the chickens were housed inreduced-pressure isolators with food and water ad lib. Mortality wasscored for 7 days after challenge.

As shown in Table 11, passive immunization of chickens with CH7-FTantiserum afforded significant protection against challenge with 3 outof 5 E. coli strains tested. Of these 3 strains against whichsignificant protection was seen, 2 strains excreted toxic activity inthe culture supernatant and one strain had toxic activity for Vero cellsonly in bacterial lysate. The 2 strains against which no significantprotection was seen both had toxic activity only in bacterial lysate.Strikingly, significant protection was only obtained against challengewith motile strains.

                                      TABLE 11                                    __________________________________________________________________________    Protection of broilers against E. coli                                        infection by passive immunization with CH7-FT                                 antiserum.                                                                                           CH7-FT                                                                              Mortality.sup.3)                                 Infection with strain  antiserum                                                                           (number out                                      No. Serotype                                                                             Toxin.sup.1)                                                                      Motile.sup.2)                                                                     Dose                                                                              administered                                                                        of total)                                                                           P < 0.05.sup.4)                            __________________________________________________________________________    CH2 O78:K80:H4                                                                           lys +   5 × 10.sup.6                                                                +      7/16 +                                          CH2 O78:K80:H4                                                                           lys +   5 × 10.sup.6                                                                -     14/16                                            CH5 O2:K1:H5                                                                             sup +   10.sup.6                                                                          +      3/16 +                                          CH5 O2:K1:H5                                                                             sup +   10.sup.6                                                                          -     6/9                                              CH6 O1:K1:H--                                                                            lys -   10.sup.7                                                                          +     15/33 -                                          CH6 O1:K1:H--                                                                            lys -   10.sup.7                                                                          -     21/36                                            CH7 O15:K14:H10                                                                          sup +   2 × 10.sup.6                                                                +      3/32 +                                          CH7 O15:K14:H10                                                                          sup +   2 × 10.sup.6                                                                -     18/29                                            CH245                                                                             O35:K--:H--                                                                          lys -   5 × 10.sup.6                                                                +      6/17 -                                          CH245                                                                             O35:K--:H--                                                                          lys -   5 × 10.sup.6                                                                -      7/19                                            __________________________________________________________________________     .sup.1) Toxic activity on Vero cells of culture supernatant (sup) or of       bacterial lysate only (lys).                                                  .sup.2) Motility of strains in Ushape tubes.                                  .sup.3) Number of dead chickens within 7 days after challenge out of          total.                                                                        .sup.4) Chisquare test for significant protection by antiserum.          

I claim:
 1. A vaccine for the protection of a bird against infection byE. coli having flagellar toxin, comprising an effective amount forprotecting a bird against E. coli flagellar toxin toxic activity ofisolated and purified E. coli toxic flagella having flagellin subunitswith a molecular weight range of about 37 to about 69 kD foundassociated in and/or with filamentous aggregates, not naturallypossessing bound carbohydrate residues, having immunizing activityagainst E. coli flagellar toxin toxic activity in birds, having toxicactivity against Vero cells and day-old chicks, and retaining toxicityon heating for 1 hour at 100° C., and a pharmaceutically acceptablecarrier.
 2. A vaccine according to claim 1, wherein the toxic activityin the flagella toxin is inactivated.
 3. A method for protecting birdsagainst an E. coli infection by E. coli having flagellar toxin,comprising administering an effective amount for protecting a birdagainst E. coli flagellar toxin toxic activity of a vaccine according toclaim
 1. 4. A method for the preparation of a vaccine for the protectionof a bird against E. coli infection comprising,(a) culturing E. colicells having flagella that possess toxic activity against Vero cells,(b) separating the flagella from the E. coli cells, and (c) adding tothe separated flagella at least one of the following:i. pharmaceuticallyacceptable salts, ii. an inactivating agent, iii. a buffer, iv. apharmaceutically acceptable carrier or diluent, v. an adjuvant, or vi.an emulsifier.
 5. A method for protecting a bird against an E. coliinfection by E. coli having flagellar toxin, comprising administering aneffective amount for protecting a bird against E. coli flagellar toxintoxic activity of a vaccine according to claim
 2. 6. The vaccine for theprotection of a bird against an Escherichia coli infection by E. colihaving flagellar toxin of claim 1, wherein the flagella have flagellinsubunits with a molecular weight of about 47 kD as measured by SDS-PAGE.7. A method for protecting a bird against an E. coli infection by E.coli having flagellar toxin, comprising administering an effectiveamount for protecting a bird against E. coli flagellar toxin toxicactivity of a vaccine according to claim 6.